This proposal outlines a general strategy in which a small molecule is used to effect the physical association of secretory pathway enzymes. Our strategy will take advantage of the modularity of glycosyltransferases in the glycolipid biosynthetic pathway as well as the high affinity interactions between the natural product, rapamycin, FK505-binding protein (FKBP12), and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin associated protein (FRAP). Utilizing chimeric glycosyltransferases, which will contain either a FRB or FKBP12 domain within them, the potential channeling of glycolipid substrates from one enzyme to the next within the biosynthetic pathway will be explored. The proposed research is comprised of a stepwise progression to our final objective: the elaboration of a small molecule whose action will heterodimerize two modified glycosyltransferases, providing a method to study potential substrate channeling and modify it. In the first aim, we will develop a set of hybrid ganglioside glycosyltransferases that contain the FKBP and FRB rapamycin binding domains and evaluate the ability of rapamycin to heterodimerize these enzymes. The second aim will utilize this controlled heterodimerization technique to investigate whether physical association of these glycosyltransferases effects correct channeling of ganglioside substrates to the next enzyme in the pathway. The third aim will apply the strategy to the selective control of the enzymes' physical association, allowing the glycolipid substrates to be channeled to specific glycosyltransferases. Thus, new unnatural ganglioside expression will be created in the cells.